Method for identifying and obtaining activated T-cells that have a demethylated Foxp3 TSDR region

ABSTRACT

This disclosure provides a method of identifying or separating activated regulatory T cells that have a demethylated Foxp3 TSDR region from a mixture of activated T cells. The mixture is depleted of cells bearing the CD154 molecule (CD40 ligand). The desired cells can be recovered using a Treg marker such as CD25, GITR, CTLA4, CD137, latent TGF-beta (LAP), GARP (LRRC32) or CD121a/b. The Treg cells having a demethylated Foxp3 TSDR region can be used, for example for preparation of pharmaceutical compositions for use in treatment.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/878,871, filed on Sep. 9, 2010 which is currently pending, which willissue on Dec. 20, 2016, as U.S. Pat. No. 9,523,076. The 0′76 patentclaims priority under 35 USC §119 to DE10-2009-040716.2 filed Sep. 10,2009. The aforelisted priority applications are hereby incorporatedherein by reference in their entirety for all purposes.

FIELD OF THE INVENTION

The invention pertains in general to the field of immunology, inparticular to the field of cell separation. The CD154 molecule (CD40ligand) is used for identifying and separating cells with a demethylatedFoxp3 TDSR region from a mixture of activated regulatory T-cells.

BACKGROUND

Specific T-cells, which actively suppress unwanted immune responses suchas, for example, to endogenous structures (auto-antigens) exist in theorganism. They are referred to as regulatory T-cells (Treg). RegulatoryT-cells are essentially involved in the life-long maintenance ofperipheral tolerance to auto-antigens. They are produced in the thymus,but the activity thereof is greatly reduced with increasing age.

Regulatory T-cells may lead to suppression of anti-tumor responses(Onizuka et al., Cancer Res. 59: 3128-3133 (1999); Shimizu et al., J.Immunol. 163:5211-5218 (1999) both incorporated by reference for allpurposes), because many tumor antigens represent classicalauto-antigens. By contrast, reduced numbers of Treg or functionalalterations in Treg may lead to autoimmune diseases during whichendogenous structures are attacked in an uncontrolled manner likeforeign substances or pathogens.

Treg are involved in maintenance of immunological self-tolerance, inthat they inhibit the activation of auto-reactive T-cells. They arecapable of suppressing both cytokine production as well as theproliferation of such potentially pathogenic T-cells. An essential stepin the identification of regulatory T-helper cells was thecharacterization of CD4+ T-helper cells, which include the constitutivealpha-chain protein of the interleukin-2 (IL-2) receptor (CD25) as asurface membrane protein.

The functional significance and the exact molecular mechanisms of thesuppression of these CD25+CD4+ regulatory T-cells and also the manner inwhich they arise have until now not been elucidated. Since the CD25receptor is also expressed by subpopulations of non-regulatory T-cells,this marker can only be used provisionally for analyzing andconcentrating Treg. CD25 cannot, however, be used particularly foridentifying and separating activated antigen specific regulatoryT-cells.

Isolation of Treg without contaminating conventional T-cells and inparticular of Treg specific for certain antigens is, nevertheless, oneof the great therapeutic aims. This is also true for the analysis ofTreg and antigen-specific Treg. (Auto)antigen-specific Treg are aspecific means for suppressing unwanted immune responses such as, forexample, autoimmune reactions in rheumatoid arthritis (RA), multiplesclerosis (MS), atherosclerosis (AS), diabetes, and psoriasis. Otherunwanted immune responses in which Treg would represent a specific meansare GvHD (graft versus host disease) in allogeneic stem celltransplantations or transplant rejection in organ transplantations (Haraet al., J. Immunol. 166: 3789-3796(2001); Taylor et al., J. Exp. Med.193: 1311-1318 (2001), both incorporated by reference for all purposes).Allergies also represent unwanted immune responses for which only a fewtherapeutic options have been available to date. A therapeutic treatmentwith Treg, which is based on the natural principal of peripheraltolerance and has been demonstrated in many experimental models wouldinvolve no side effects by comparison with conventionalimmunosuppressive medicaments and would be curative.

In relation to the stated importance of Treg, the molecules andmechanisms involved in the suppression, and reliable Treg markers, areof great importance. A molecular biological Treg marker, thetranscription repressor FoxP3, which belongs to the Forkhead-family, isacknowledged in the art (Hori et al (2003) Science 99(5609): 1057-61,incorporated by reference for all purposes). In addition, regulatoryT-cells can be identified on the bases of expression of the CD25molecule (Thornton and Shevach (1998) J. Exp. Med. 188 287-296;Sakaguchi et al. (1995) J. Immunol. 155 1151-1164, both incorporated byreference for all purposes).

To date, however, it has not been possible to identify and isolate thoseTreg cells, which recognize a specific antigen. This would be possibleif specifically activated Treg could be separated. This would be aprerequisite for specific therapies with Treg, for example, forautoimmune diseases in which the autoimmune reactions underlying thedisease should be suppressed, but not those immune reactions launchedagainst tumor cells.

According to Choi et al. (2004, J. Leukocyte Biology 76:1-7,incorporated by reference for all purposes), no evidence exists thatCD137 (4-1 BB) can be employed as a discriminative marker for Tregversus CD25. In an international application (WO 2007/110249,incorporated by reference for all purposes), it is described that Tregselectively express CD137 at about 4 hours after activation, whereasconventional T-cells only start to express CD137 after about 12 to 16hours. Although this early time window can be used to isolate regulatoryT-cells only a few hours after activation, this method is prone toerrors and therefore is of little practical use. A strong variability inthe activation state of the T-cells after obtaining them from blood ortissues as well as the variability of the special kinetics of CD137 onthe individual T-cells leads to a contamination of the regulatoryT-cells with conventional T-cells, which can be small or large,depending on the time point of the isolation, and which limits theusability of isolated cells dramatically. By using an early time pointfor separating the cells, the contamination can be kept small, althoughthe yield of the isolated regulatory T-cells is grossly decreased, sincenot all regulatory T-cells have yet become positive for CD137. As thenumber of regulatory T-cells, in particular of antigen-specificregulatory T-cells is small to begin with, the decrease in yielddiminishes the usability of the method, since many therapeutic uses canno longer be applied.

Therefore, to date, it has not been possible to identify and separatethe contaminating effector cells (e.g. FoxP3 negative and/or cytokinepositive) from the regulatory T-cells (defined by the expression ofknown markers, in particular CD25, CD25+CD127−, GITR+) or todifferentiate antigen-activated regulatory T-cells from activatedconventional T-cells.

The term “conventional T cells” as used herein refers to all T cellswhich do not belong to the “naturally occurring” regulatory T cellswhich are characterized by stable expression of the transcription factorFoxp3+ and which typically express CD25 constitutively and no or lowlevels of CD127. Conventional T cells especially refer to T cells whichexert immune activating effector functions, i.e. production of effectorcytokines such as IL-2, IFN-gamma, IL-4, IL-5, IL-9, IL-17, IL-22.

It has been difficult to identify and/or isolate regulatory T-cells.Markers described thus far do not allow the possibility of identifyingthe totality of regulatory T-cells, because not all regulatory T-cellsexpressed defined specific markers. Accordingly, it is only possible toidentify and/or separate subpopulations, such as, for example only theTreg, which strongly expresses the CD25 receptor. Furthermore, variousother cell subpopulations that have the same cell surface markers (suchas CD4 for Th1 and Th2, or other Th-cells or CD25 for activated T-cellsor B-cells) cannot be distinguished from regulatory T-cells.

In particular, it has not been possible to identify activated Treg afterstimulation with defined antigens by means of specific activationmarkers. Although activation of regulatory Th-cells leads to enhancedexpression of CD25 and CD38, they are expressed like all other describedT-cell activation markers also in other T-cells, so that it has not beenpossible to separate any antigen-specific Treg by means of specificactivation markers.

Frentsch et al. (2005, Nat Med. 11(10):1118-24) and the application EP 1420 253, both incorporated by reference for all purposes, disclose thatCD154 (CD40 ligand) is expressed on all activated T-cells, wherein nodistinction is made by the authors between conventional and regulatoryT-cells regarding expression of CD154. It was not known whether CD154can be used to discriminate between activated conventional and activatedregulatory T-cells.

Rausch et al (2008, Infection and Immunity, 76(5):1908-1919,incorporated by reference for all purposes) disclose that they were ableto identify murine antigen-specific Teff and Treg cells by theexpression of CD154. The authors in fact assumed that CD154 is alsoexpressed by antigen-activated Treg and could be used for positiveselection of antigen-activated Treg.

These murine data do not suggest using CD154 as discriminative markerbetween activated conventional and activated regulatory T-cells.

Most of the methods used to identify and/or isolate regulatory Th-cellson the basis of the expression of a particular cell surface markerdepend on recognition of a marker and binding of an antibody. When it isnot possible to use a single marker to identify and/or isolate aparticular cell type, it is necessary to find a combination of markersand the cognate antibodies (Levings et al., J Exp. Med. 193 (11):1295-1302 (2001). Such experiments may in practice be very complicatedand difficult to carry out. Moreover, it is possible that the binding ofthe antibodies influences the activity of the target cell or theexpression of other markers, thus having a negative influence on theidentification and/or separation process with the other antibodies.

To date, no specific identification and/or separation of exclusivelyliving regulatory T-cells has been carried out. The only specific markerdescribed to date, FoxP3, may moreover, according to recentinvestigations, be induced also in non-regulatory T-cells (Fontenot J.D. 2003; Hori S. et al. 2003. Since FoxP3 is present as an intracellularprotein, it is further not possible according to the present state ofthe art to identify and/or isolate living regulatory cells, for example,on the basis of a FoxP3 antibody. Again, the problem that arises is thatonly the totality of the Treg can be identified, but not Treg specificfor pre-defined antigens. A reliable identification and/or separation ofthe Treg and in particular of antigen-specific Treg have therefore notbeen possible.

BD Pharmingen TM: “Technical Data Sheet: PE conjugated mouse anti-humanCD137 (4-1 BB)”, May 7, 2005, also discloses that the CD137 markerrepresents an activation marker for all T-cells. The application WO2005/124346, describes CD137 as being a marker on CD4⁺CD25⁺; however,the publication does not disclose that CD4⁺CD25⁺ especially express at aparticular time during activation of the CD137 marker as compared toCD4⁺ CD25⁻ T-cells. The authors of WO 2005/124346 disclose, on the otherhand, that both cell populations of CD4⁺ CD25⁺ and CD4⁺ CD25⁻ T-cellsexpress the CD137 marker to the same extent after activation thereof.The documents mentioned represent the opinion in the art that the CD137marker cannot be used as a discriminatory marker for CD25⁺ Treg-cellsversus CD25⁻ T-cells. In particular, BD Pharmingen TM describes aPE-conjugated mouse anti-human CD137 antibody. WO 2005/124346 disclosesthe co-stimulation of freshly isolated CD4⁺ CD25⁺ mouse Treg-cells viaCD137. The Treg-cells are not stimulated via CD137, but through thepresence of strong acting stimuli such as CD3. Most of the so-calledfresh Treg-cells do not express CD137. For example, it is shown in FIG.5 of WO 2005/124346 that co-stimulation with 4-1BBL exerts activatingproperties in particular also on CD25⁻ cells. It is disclosed in FIG. 1din an unambiguous fashion that CD4⁺ CD25⁺ and CD4⁺ CD25⁻ T-cells bothexpress CD137 during activation, so that the publications mentioned donot motivate the skilled artisan to use CD137 for specific presentationor isolation of CD4⁺ CD25⁺ Treg-cells from cell mixtures.

WO 2007/110249 is disclosed that Treg express CD137 selectively withinan early time frame after activation (only after about 4 hours), whereasconventional T-cells begin to express CD137 after about 12 to 16 hours.Although this time frame may be used for identifying activatedregulatory T-cells, the variability of the expression kinetics as wellas the short stimulation time leads to a massive decrease of cell purityand yield. Before the invention disclosed and claimed below, there hasbeen no method available for enrichment of activated Treg cells allowingthe simultaneous depletion of activated conventional T cells and Foxp3+T cells with instable Foxp3 expression.

SUMMARY OF THE INVENTION

The invention relates to a method of identifying and separatingnon-regulatory T-cells (conventional T-cells) from a mixture comprisingregulatory T-cells by using of the CD154 molecule (CD40 ligand) throughdepletion of CD154+ T-cells from the mixture or in combination withadditional positive selection of Treg using markers that are specificfor regulatory T-cells, such as for example, CD25, GITR, CTLA4 ormarkers which are specific for activated regulatory T-cells, such as,for example, CD137, “latent TGF-beta (LAP)”, GARP (LRRC32), CD121a/b.The invention relates also to a kit comprising an antibody for detectingCD154 and at least one additional antibody for detecting markers foractivated or non-activated regulatory T-cells. The antibodies can becoupled to a fluorescent dye or magnetic microparticles. The regulatoryT-cells and the conventional T-cells can be activated with eitherpolyclonal or specific antigens. The activation can be performed invitro or in vivo and can be performed on un-separated T-cells orpre-selected regulatory T-cells, for example, using CD25, CTLA-4(CD152), GITR or other typical markers for regulatory T-cells.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 presents eight different scatter plots from analysis of cells byfluorescence activated cell sorting. It shows that shortly afterstimulation a large fraction of the CD137+ Treg also expressed CD154.

FIGS. 2A and 2B show that within the CD137+ T-cells mixture CD154T-cells express more FoxP3.

FIG. 3 shows that within the CD137+ T-cell mixture only CD154+ T-cellsexpress effector cytokines.

FIG. 4 shows the time window for sorting and reanalysis of CD137/CD154double- and single-positive T-cells.

FIG. 5 shows that only CD154− Treg cells show suppressive properties.

FIGS. 6A and 6B shows that CD25++ FoxP3+ Treg contain CD154+ T-cells.

FIGS. 7A, 7B and 7C show that Helios is almost exclusively expressed byCD137+CD154− Treg.

FIG. 8 shows that CD137+CD154− Treg show strong de-methylation of theFoxP3 promoter.

FIG. 9 shows that CD137 and CD154 are also expressed on expanded Tregfrom various populations.

FIG. 10A shows that depletion of CD154+ Treg from a Treg expansionculture increases the FoxP3 purity and the long terms stability of FoxP3expression. FIG. 10B shows FoxP3 purity after separation via CD154.

DETAILED DESCRIPTION OF THE INVENTION

This disclosure provides technology for removing living, activatedconventional T-cells from mixtures comprising regulatory T-cells,wherein CD154 can be used as a marker and wherein CD154-specificantibodies can be used to identify and/or remove activated conventionalT-cells. By combining the depletion of conventional T-cells with aseparation step (preceding or consecutive) in which regulatory T-cellsare identified and enriched (either using conventional markers fornon-activated regulatory T-cells, like CD25, CTLA4 or GITR, or throughlabeling them with a marker specific for activated Treg like CD137,latent TGF-beta (LAP), GARP (LRRC32), CD121a/b), the purity and yield ofthe activated regulatory T-cells can be largely increased, which is aprerequisite for many therapeutic and diagnostic applications.

As used herein, the terms “depletion”, “depleting” and the like, in thecontext of cell isolation, purification or enrichment, have the normalmeaning in the art, and refer to removal of specified cells (e.g.,CD154+ cells) from a mixture or starting population of cells. Method fordepletion are well known in the art and are described herein, andinclude, for example, FACS or MACs sorting in which specified cells in apopulation (e.g., CD154+ cells) are labeled and removed from thepopulation of cells resulting in a new population in which the specifiedcells are absent or present in a lower proportion than in the startingpopulation. It will be recognized that “depletion” does not require thatthe specified cells be entirely removed or that the new population beentirely free of the specified cells. Typically, depleting specifiedcells from a population means reducing the representation of such cells(measured as a percentage of all of the cells in the population) by atleast about 50%, at least about 60%, at least about 70%, at least about80%, at least about 90%, at least about 95%, at least about 98%, or atleast about 99%).

As used herein, the term “positive selection”, in the context of cellisolation, purification or enrichment, has the normal meaning in theart, and refers to the enrichment of specified cells (e.g., CD137+cells) from a mixture or starting population of cells. Methods forpositive selection are well known in the art and are described herein,and include, for example, FACS or MACS sorting in which specified cellsin a population (e.g., CD137+ cells) are labelled and isolated from thepopulation of cells in that the isolated cells result in a newpopulation in which the specified cells are present in a higherproportion than in the starting population.

Therefore, the invention refers to the teaching that activated, and inparticular, antigen specifically activated conventional T-cells can beseparated from CD4⁺CD25⁺ Treg that also have been activated by using themarker CD154 through depletion of the CD154+ cells from the mixture,since activated Treg express no CD154.

Additionally, regulatory cells can be positively identified and/orseparated through the expression of one and/or several markers. Forthis, all markers known to a person of skill in the art for identifyingand/or separating Treg from activated Treg can be used. Preferably, themarkers CD137, CD25, CTLA-4 (cytotoxic T lymphocyte antigen-4), GITR(glucocorticoid-induced TNF receptor), FoxP3, IL-10, CD69, ICOS, OX40and TGF-beta can be used alone or in combination. For this, all markersknown to a person of skill in the art for excluding or depletingnon-regulatory cells can be used in combination. Particularly preferredis the use of CD137 as a marker for living, activated regulatory cells.Either activated regulatory T-cells are directly identified orseparated, or the activation is induced through additional cells,proteins, peptides, pathogens or other substances.

The method can also be used for enrichment of activated regulatoryT-cells in a mixture of cells.

One embodiment of the method is the separating of activated regulatoryT-cells from a mixture comprising activated regulatory T-cells andactivated conventional T-cells by contacting the cell mixture with amolecule binding CD154 and depletion of CD154+ T-cells from the mixtureobtaining a population of activated regulatory T-cells.

Another embodiment of the method is the separating of activatedregulatory T-cells from a mixture comprising activated regulatoryT-cells and activated conventional T-cells, the method comprising thesteps:

a) contacting the cell mixture with

-   -   i. a molecule binding CD154 and depletion of CD154+ T-cells from        the mixture; or    -   ii. a molecule binding a marker that is specific for regulatory        T-cells or that is specific for activated regulatory T-cells and        positive selection of the cells that bind to said binding        molecule;

b) contacting

-   -   i. the cells of step a) i. with a molecule binding a marker that        is specific for regulatory T-cells or that is specific for        activated regulatory T-cells and positive selection of the cells        that bind to said binding molecule obtaining a population of        activated regulatory T-cells; or    -   ii. the cells of step a) ii. with a molecule binding CD154 and        depletion of CD154+ T-cells obtaining a population of activated        regulatory T-cells.

The invention relates to T-cells from mammals, in particular, humans,mice, rats, rabbits or dogs. The DNA and protein sequences of themolecules referred to here are known to a person of skill in the art andare publicly available in databases. When necessary, the sequences canalso be obtained using routine laboratory techniques.

The invention also relates to the use of CD154 as well as its homologuesand fragments. The homologues may show, for example, a homology of atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, at least 98%, at least 99% to human CD154. The fragments ofCD154 are without limitation, i.e. each fragment of naturally occurringCD154 molecule can be used according to the invention. Antibodies, forexample, only bind to a part of the CD154 molecule.

The invention refers to both in vitro and in vivo use.

In a further aspect, the invention refers to use of CD154 as a negativeselection marker greatly increases the window of time for the use of themarker CD137 for the positive selection of Treg and that 2 to 24 hoursafter activation, regulatory T-cells can be isolated with great purityand yield.

The separated cells can be collected in a suitable container, forexample, a collection tube that allows for the survival and/or growth ofthe cells. Different media are commercially available and can be usedbased on the need of the type of cells identified and/or separated. Suchmedia can be, e.g. DMEM, HBSS, DPBS, RPMI, Iscove's medium, X-VIVO™medium, etc., which may be often supplemented with fetal calf serum,human serum or serum substitutes.

The identified and/or separated cell populations can be used immediatelyor can be cultivated in vitro after isolation. Subsequently, the cellscan be frozen or they can be frozen before being separated. In case thecells are to be stored for a longer period of time it is preferred to doso at around −80° C. or in liquid nitrogen in order to in ensure thatthe cells can be used again after thawing. For this purpose, the cellsare normally stored in DMSO and/or FCS/HS together with a medium,glucose etc. Immediately after the cells have been thawed, the cells caneither be used directly for therapeutic purposes or in in vitroexperiments or they can be expanded and/or differentiated using growthfactors, antigens, cells etc.

The regulatory Th-cells are identified and/or separated from cellssamples from mammals, in particular from humans and preferably frompatients. The cell samples can be derived, for example, from bloodsamples containing immune cells (body fluids like peritoneal,cerebral-spinal, pleural liquid, and/or synovial fluid), lavage liquidsfrom hollow organs (airways, lungs), homogenates and aspirates of lymphnodes, spleen, tonsils and/or other lymphatic tissue. The cells can beremoved from animal tissues (for example from the spleen or the lymphnode of an animal). The cells may also be a part of a blood sample, forexample from a subject or a human patient, particularly preferred from aperipheral blood sample.

Activated regulatory Th-cells are directly able to suppress the activityof T-cells. It has been shown in animal models that activated regulatoryT-cells can prevent the development of autoimmune diseases, oftransplant rejection reactions, and allergic reactions. It is assumed inanalogy thereto that activated regulatory T-cells can also suppress thedevelopment of chronic immune reactions in humans. By using the CD137marker, activated regulatory Th-cells are specifically identified and/orseparated. Preferably, in vitro activated regulatory Th-cells with thephenotype CD4+CD25+ FoxP3+ are previously identified and/or separated.The activation occurs preferably using the specific antigens, e.g.peptides, proteins, or pathogens or cells or polyclonal stimuli, e.g.chemical compounds like PMA/ionomycin, superantigens like SEB or PHA orstimulatory antibodies like anti CD3 (or mixtures thereof).

By using the CD154 marker, activated conventional Th-cells canpreferably be removed from mixtures comprising regulatory T-cells, inparticular, when additional selection methods using markers foractivated/non-activated regulatory cells (CD25, CD137) are usedsimultaneously or subsequently, which allows for a preferred used of theinvention for identifying isolating antigen-specific regulatoryTh-cells.

After obtaining a cell mixture from a patient and/or a subject,regulatory T-cells can be enriched using, for example, CD25. These cellscan be subsequently stimulated with a particular antigen. For thispurpose, antibodies, peptides, proteins, chemicals substances (ormixtures thereof), or pathogens or cells may be used. After a particularactivation time, the regulatory Th-cells are identified and/orseparated, for example, through the use of the CD137 marker. Thereby,antigen-specific regulatory Th-cells are preferably identified and/orseparated.

The cells, which are separated according to the invention, usuallyreflect the immunological status of the donor regarding the number, theorigin, and the T-cell antigen receptor specificity of the Treg cell.This information may be used in diagnostics referring to immunologicaldisorders, for example, cancer-related immunosuppression, autoimmunedisorders, atopical states.

Further, Th-cells of the patient can be contacted in vitro with cells,cell extract separated antigens or proteins of an organ donor before orafter the transplantation. Subsequently, the activated regulatoryTh-cells are identified and/or separated using the CD137 marker after asuitable activation period.

The cells can be identified and/or separated according to all methodsknown to the person skilled in the art. Preferred for the identificationof cells are in particular cell sorting (e.g. magnetic cell sorting(MACS)), fluorescence activated cell sorting (FACS), ELISA, PCR and/orall fluorescence microscopes known in the art. Particularly preferred isthe use of flow-cytometry (FACS). For the separation of the cells, thosecell separation systems in which the cells are sorted using a magneticfield (e.g. MACS) or through flow-cytometry (FACS) are particularlypreferred. Further, it is preferred the expression of CD137 is measuredafter stimulation.

The regulatory Th-cells are stimulated by substances known to a personof skill in the art. The expression of CD137 is measured 1 to 18 hoursafter stimulation. Preferably, the expression is measured 3 to 8 hoursafter the stimulation.

According to the invention, CD154 is a specific marker for activatedconventional T-cells within a time frame of about 0 to 18 hours afteractivation, preferred within about 0 to 12 hours, particularly preferredwithin about 0 to 10 hours, especially preferred within about 1 to 8hours, most referred within about 3 to 8 hours, in particular within 4hours.

The preferred optimal time point for separation is the time point atwhich a maximum number of the activated regulatory T-cells express CD137and at which a minimum number of non-regulatory cells express CD137.This time point can be determined through an analysis of the CD137expression at different time point after stimulation.

In a further preferred embodiment, the regulatory Th-cells areidentified and/or separated after a stimulation of the cells in the cellmixture.

The cells can be stimulated by antigens, proteins, peptides, chemicalsubstances, cells, growth factors, antibodies and/or ligands.

The culture may also contain suitable growth factors. Growth factors aredefined as molecules that simulate survival, growth, and/ordifferentiation of a cell either in a culture or within a tissue byinteraction with a transmembrane receptor. Growth factors includepolypeptides and non-polypeptide factors. Particular growth factors thatcan be used for culturing the separated and/or used cells include theinterleukins, for example, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17,IL-18, IL-21, IL-22, IL-23, IL-27 etc.; antigens, for example, peptideantigens, protein antigens, like for example alloantigens, preferablytogether with antigen presenting cells, lectins, for example ConA,[Alpha]-CD3, LPs, etc.

The culture may also contain antibodies or specific ligands (purifiedligands, FC fusion proteins or other recombinant forms of the “leucinezipper”) for cell surface receptors that may stimulate or inhibit Tregactivity. Antibodies or ligands that bind TNFR or other co-stimulatingmolecules on Treg and stimulate and/or increase Treg activity, thatcounteract Treg activity (and cause cell division) or that simulateapoptosis of Treg may also be present in the media. Normally, thespecific culture parameters serve a particular purpose, for example themaintenance of Treg cell activity, etc.

As used herein, the term “antibody” is intended to include polyclonaland monoclonal antibodies, chimeric antibodies, haptens and antibodyfragments, and molecules which are antibody equivalents in that theyspecifically bind to an epitope on the product antigen. The term“antibody” includes polyclonal and monoclonal antibodies of any isotype(IgA, IgG, IgE, IgD, IgM), or an antigen-binding portion thereof,including, but not limited to, F(ab) and Fv fragments such as sc Fv,single chain antibodies, chimeric antibodies, humanized antibodies, anda Fab expression library.

One goal is to have a sufficient concentration of antibodies in thereaction mixture such that the efficiency of separation is not limitedby too few antibodies. The necessary concentration may be determinedthrough titration.

The method for separating the cells can be any method that maintains theactivity of the cells. A preferred method is a phosphate buffered saltsolution containing 0.1 to 0.5% BSA or in equal amounts autologouspooled serum or substitute serum substances. Various media arecommercially available and may be used depending on the cell type andthe experiments that are to be performed. Available media are forexample, Dulbecco's modified Eagle's Media (DMEM), Hank's Basic SaltSolution (HBSS), Dulbecco's Phosphate buffered salt solution (DPBS),RPMI, Iscove's Media, X-VIVO™ PBS with 5 mM EDTA etc. often supplementedwith fetal calf serum BSA, HSA etc.

The labeled conventional cells are then separated depending on theexpression of CD154. The regulatory cells that are not separated can becollected by any method that maintain the vitality of the cells.Normally, the cells are being collected in a collection tube with serum.Various media are commercially available and can be used depending onthe identified and/or separated cells, for example, dMEM, HBSS includingdPBS, RPMI, Iscove's Media etc., often supplemented with fetal calfserum, autologous or pooled serum or serum substitutes.

It is further preferred that the antigens for detecting CD137 arecoupled with a fluorescent dye or to magnetic microparticles.

The antibody is directly or indirectly conjugated to a magnetic reagent,such as a superparamagnetic microparticle (microparticle). Directconjugation to a magnetic particle is achieved by use of variouschemical linking groups, as known in the art.

Alternatively, the antibody is indirectly coupled to the magneticparticles. The antibody is directly conjugated to a hapten, andhapten-specific, second stage antibodies are conjugated to theparticles. Suitable haptens include digoxin, digoxigenin, FITC,dinitrophenyl, nitrophenyl, avidin, biotin, etc. Methods for conjugationof the hapten to a protein, i.e. are known in the art, and kits for suchconjugations are commercially available.

For flow-cytometry, e.g. FACS (fluorescence activated cell sorting), andthe magnetic cell sorting (e.g. MACS) antibodies are used that may becoupled with fluorescence markers or magnetic microparticles that areknown to a person of skill in the art, like FITC, phycoerythrin (PE),allophycocyanin (APC), cascade yellow and peridinin chlorophyll protein(PerCP). The antibodies may also be coupled to haptens and may then bedetected with hapten-specific secondary antibodies. A combination ofantibodies can be used to detect, analyze and/or isolate various cellswith different properties on the basis of markers, like secretedproteins or characteristic surface molecules. In particular,fluorescence labeled antibodies can be used for FoxP3, CD25 and/or CD4markers. It is particularly preferred to use CD137 antibodies that arecoupled to fluorescence dye or to magnetic microparticles. Magneticmicroparticles may be used directly or indirectly (hapten for examplebiotin and anti-hapten microparticle of fluorochrome, e.g. PE or APC andanti-fluorochrome microparticles).

In another preferred embodiment, the regulatory Th-cells are directlyidentified and/or separated or isolated from blood, peripheralmononuclear blood cells (PBMC), body tissue or cells from tissue fluids.

The regulatory Th cells are normally identified and/or separated fromcell samples from mammals, but especially from humans and preferablyfrom test subject and/or patients. The regulatory Th cells may bederived for example from blood samples which comprise immune cells(peritoneal, cerebral-spinal, pleural and/or synovial body fluids,lavage fluids from hollow organs (airways, lungs), homogenates andaspirates of lymph nodes, spleen, tonsils and/or other lymphatic tissue.The regulatory Th cells may also be obtained from animal tissues (e.g.from the spleen or the lymph nodes of an animal). The Th cells may alsobe part of a blood sample, e.g. from a test subject or human patient,particularly preferably from peripheral mononuclear blood cells (PBMC),body tissue or cells of tissue fluid.

Also provided are kits for practicing the methods of the invention. Kitsmay comprise a package with at least one container (e.g., vial)comprising an antibody for detecting CD154 for separating activatedconventional T cells and instructions for such separation, as well assuitable buffers, labels, etc. The kit may also include reagents forseparating an activated or non-activated regulatory Th cell based onantigens expressed by the cells, a number of examples of which aredescribed herein (e.g. CD137, CD25). In some embodiments the marker isspecific for regulatory T-cells (e.g., CD25+, GITR+ and CD127−). In someembodiments the marker that is specific for activated regulatory T-cells(e.g., CD137, latent TGF-beta (LAP), GARP (LRRC32) and CD121a/b). Insome embodiments the kit comprises a first container containinganti-CD154 and at least 1, at least 2, or at least 3 additionalcontainers each containing an antibody that binds T-cells. In someembodiments each of the antibodies in the kit is differently labeled sothat they can be distinguished.

It is preferred for the CD154 antibody in the kit to be coupled to afluorescent substance, to a hapten or to magnetic microparticles.

A 4-1BB receptor antibody which is coupled to a fluorescent marker, ahapten or magnetic microparticles is provided for identifying and/orseparating the activated, live regulatory Th cells. Fluorescent markersare known to the skilled worker, such as, for example, FITC,phycoerythrin (PE), allophycocyanin (APC), cascade yellow and peridininchlorophyll protein (PerCP).

It is possible through use of the kit comprising a CD154 antibodycoupled to a fluorescent marker, haptens or magnetic microparticles toidentify and/or separate activated regulatory Th cells. Particularlypreferred are the identification and/or separation of live, activatedregulatory Th cells.

In this embodiment, the method of the invention can be used for exampleto identify and/or separate Th cells from a blood sample (or from ablood derivative) or from cell mixture derivatives of lymphatic organsor tumors of mammals.

The separated activated regulatory Th cells can be used before and/orafter cloning and/or growing and/or concentrated in cell mixtures inand/or as pharmaceutical composition in the therapy or prevention ofdiseases. It is additionally possible for the coding gene sequences ofthe TCR (T-cell receptor) to be isolated from the separated regulatory Tcells and be used for further therapeutic purposes such as, for example,for cellular therapies. It is additionally possible to employ theactivated regulatory Th cells in the form mentioned in furtherinvestigations and/or analyses. The pharmaceutical composition can beused for the treatment and/or prevention of diseases in mammals,possibly including administration of a pharmaceutically effective amountof the pharmaceutical composition to the mammal.

The disease may be any disease, which can be treated and/or preventedthrough the presence of a separated cell and/or through increasing theconcentration of the relevant cells in/at the relevant place, or inwhole mammalian subjects and/or patients. The cell may be for example anactivated regulatory Th cell, and the treated and/or preventivelytreated disease may be an autoimmune disease, an infectious disease, anallergy, transplant versus host disease (or allogeneic transplantrejection) and/or any other disease initiated by hypersensitivity.

Diseases for which the use set forth in the invention is particularlysuitable are those arising through and/or during a lack of regulation ofthe immune response. These diseases may be transplant rejections,allergic conditions, certain infectious diseases and/or autoimmunediseases.

It is known in connection with autoimmune diseases that the immunesystem attacks endogenous structures, such as, for example, inrheumatoid arthritis, insulin-dependent diabetes mellitus (IDDM),multiple sclerosis (MS), atherosclerosis, psoriasis, allogeneic stemcell transplantation, organ transplantation. A therapy with specificactivated regulatory T cells is advantageous here. The goal inneoplastic diseases should be to eliminate regulatory T cells specificfor tumor antigens so that the immune system can initiate efficientanti-tumor immune responses.

The invention also relates to the use of a composition of the inventionsuch as a pharmaceutical composition for the treatment of diseases whichare associated with a deficiency of cellular immunity, for example inthe defect according to ICDIO code: D.84.4. Possibilities in thisconnection are septic disorders, inflammatory reactions and fever,autoimmune diseases and diseases of impairment of cell division such as,for example, cancer.

Accordingly, the invention also pertains to a method for treating one ofthe diseases mentioned herein using a highly pure population ofactivated Treg, as well as a method for preparing a medicamentcomprising a highly pure population of activated Treg for treating suchdiseases, as well as the use of a highly pure population of Treg fortreating such diseases.

Inflammations in the sense of the invention are the reaction, borne bythe connective tissue and the blood vessels, of the body to anexternally or internally induced inflammatory stimulus with the purposeof eliminating or inactivating it and repairing the stimulus-relatedtissue damage. Inducing effects can be exerted by mechanical stimuli(foreign bodies, pressure, injury) and other physical factors (ionizingradiation, UV light, heat, cold), chemical substances (alkalis, acids,heavy metals, bacterial toxins, allergens and immune complexes) andpathogens (microorganisms, worms, insects) and pathological metabolicproducts, dysfunctional enzymes, malignant tumors. The event starts witha brief arteriolar constriction (through the action of adrenaline) withinadequate blood flow and tissue alteration, followed by the developmentof the classical local signs of inflammation (cardinal symptoms;according to GALEN and CELSUS), i.e. of redness (=rubor; dilation ofvessels due to histamine), warmth (=calor; through a local increase inmetabolism), swelling (=tumor; through escape of protein-rich liquidfrom the vessel walls which are altered-inter alia by histamine-,assisted by the slowed circulation of blood in the sense of prestasis tostasis), pain (=dolor; as a consequence of the increased tissue tensionand pain-inducing inflammatory products, for example bradykinin) andfunctional impairment (=functio laesa). The process is supplemented bydisturbance of the electrolyte balance (trans-mineralization), invasionof neutrophilic granulocytes and monocytes through the vessel walls (seealso leukotaxis), the latter with the purpose of eliminating theinflammatory stimulus and damage to necrotic cells (phagocytosis); thereis also invasion of lymphocyte effector cells which lead to theproduction of specific antibodies against the inflammatory stimulus(immune response), and eosinophils (in the healing phase or—veryearly—in the allergic-hyperergic event). The activation of thecomplement system brought about by the reaction releases fragments (C3aand C5a) of this system which—like histamine and bradykinin—act asmediators of the inflammation, specifically in the sense of stimulatingthe chemotaxis of the cited blood cells; there is also activation ofblood clotting. This is followed by damage (dystrophy and coagulationnecrosis) of the relevant organ parenchyma. The whole body respondsdepending on the intensity and nature of the inflammation with fever,stress, leucocytosis and alterations in the composition of plasmaproteins (acute-phase reaction), which lead to an increased erythrocytesedimentation rate. Preferred inflammations in the sense of theinvention are the purulent, the exudative, the fibrinous, thegangrenous, the granulomatous, the hemorrhagic, the catarrhal, thenecrotic, the proliferative or productive, the pseudomembranous, theserous, the specific or the ulcerative inflammations.

Autoimmune diseases in the sense of the invention are diseases partlyattributable to the production of auto-antibodies and their harmfuleffect on the whole body or organ systems, i.e. auto-aggression.Autoimmune diseases may on the other hand also be diseases in which Tcells possess a predominant role in the pathogenesis or initiation.Classification as organ-specific, intermediary and/or systemicautoimmune diseases is possible. Preferred organ-specific autoimmunediseases are Hashimoto's thyroiditis, primary myxedema, thyrotoxicosis(Basedow's disease), pernicious anemia, Addison's disease, myastheniagravis and/or juvenile diabetes mellitus. Preferred intermediaryautoimmune diseases are Goodpasture's syndrome, autoimmune hemolyticanemia, autoimmune leucopenia, idiopathic thrombocytopenia, pemphigusvulgaris, sympathetic ophthalmia, primary biliary cirrhosis, autoimmunehepatitis, ulcerative colitis and/or Sjogren's syndrome. Preferredsystemic autoimmune diseases are rheumatoid arthritis, rheumatic fever,systemic lupus erythematosus, dermatomyositis/polymyositis, progressivesystemic sclerosis, Wegener's granulomatosis, panarteritis nodosa and/orhypersensitivity angiitis. Typical autoimmune diseases arethyrotoxicosis, thyroid-related myxedema, Hashimoto's thyroiditis,generalized endocrinopathy, pernicious anemia, chronic gastritis of typeA, diseases of single or all corpuscular elements of the blood (forexample autoimmune hemolytic anemia, idiopath. thrombocytopenia or-pathy; idiopath. leucopenia or agranulocytosis), pemphigus vulgaris andpemphigoid, sympathetic ophthalmia and some types of uveitis, primarybiliary cirrhosis of the liver and chronic aggressive autoimmunehepatitis, diabetes mellitus of type I, Crohn's disease and ulcerativecolitis, Sjogren's syndrome, Addison's disease, lupus erythematosusdisseminatus and as discoid form of this disease, as dermatomyositis andscleroderma, rheumatoid arthritis (=primary chronic polyarthritis),antiglomerular basement membrane nephritis. An aggressive immuneresponse as a result of collapse of immunotolerance of self-determinantsand a preponderance of inflammatory T-helper cells are the basis. Theproduction of autoantigens is also possible, for example through hostproteins combining with haptens (for example drugs), through ontogenetictissue which develops only after development of self-tolerance and forprotein components unmasked through changes in the conformation of theproteins in connection for example with infection by viruses orbacteria; also for new proteins which have arisen in connection withneoplasms first.

Septic diseases in the sense of the invention are disorders resultingfrom continuous or periodic invasion of pathogenic bacteria and/or theirtoxins from a disease focus and spread thereof via the lymph and bloodto general or local infection.

Sepsis in the sense of the invention are preferably wound sepsis(phlegmone, thrombophlebitis, lymphangitis), puerperal sepsis (inpuerperal fever), otogenic sepsis (in otitis media), tonsillogenicsepsis (in angina, peritonsilitis), cholangitic sepsis (in purulentcholecystitis, cholangitis), pylephlebitic sepsis (in pylephlebitis),umbilical sepsis (in omphalitis etc.), urosepsis and in dentalgranuloma. Sepsis in the sense of the invention may occur acutely tohighly acutely (foudroyantly), subacutely (for example as endocarditislenta) or chronically, but of course also as neonatal sepsis. Sepsis inthe sense of the invention are therefore all pathogenic alterations in apatient which may be associated with intermittent fever and chills, andwith spleen tumor, with toxic reactions or damage of the bone marrow orblood (polynuclear leukocytosis, anemia, hemolysis, thrombocytopenia) orelse with pathogenic reactions on the heart and vasomotor nerves(tachycardia, centralization of the circulation, edemas, oliguria;possibly shock) or of the digestive tract (dry, coated tongue,diarrheas) or else with septicopyemia (pyemia with formation of septicinfarctions and metastatic abscess).

Disorders associated with a deficiency of the cellular immune systeminclude: AIDS, acne, albuminuria (proteinuria), alcohol withdrawalsyndrome, allergies, alopecia (hair loss), ALS (amyotrophic lateralsclerosis), Alzheimer's disease, AMD (age-related macular degeneration),anemia, anxiety disorders, anthrax, aortic sclerosis, arterial occlusivedisease, arterial calcification, arterial occlusion, temporal arteritis,arteriosclerosis, arteriovenous fistulas, arthritis, arthrosis, asthma,respiratory failure, autoimmune disease, AV block, acidosis, prolapseddisc, peritonitis, pancreatic cancer, Becker's muscular dystrophy,benign prostatic hyperplasia (BPH), bladder carcinoma, hemophiliac,bronchial carcinoma, breast cancer, BSE, Budd-Chiari syndrome, bulimianervosa, bursitis, Byler syndrome, bypass, chlamydial infection, chronicpain, cirrhosis, concussion, Creutzfeld-Jakob disease, bowel carcinoma,bowel cancer, intestinal tuberculosis, depression, diabetes insipidus,diabetes mellitus, diabetes mellitus juvenilis, diabetic retinopathy,Duchenne's muscular dystrophy, duodenal carcinoma, progressive musculardystrophy, dystrophy, ebola, eczema, erectile dysfunction, obesity,fibrosis, cervical cancer, uterine cancer, cerebral hemorrhage,encephalitis, hair loss, hemolytic anemia, hemophilia, pet allergy(animal hair allergy), skin cancer, herpes zoster, myocardialinfarction, heart failure, inflammation of heart valves, cerebralmetastasis, stroke, brain tumor, testicular cancer, ischemia, Kahler'sdisease (plasmocytoma), infantile paralysis (poliomyelitis), boneatrophy, contact eczema, paralysis, cirrhosis of the liver, leukemia,pulmonary fibrosis, lung cancer, pulmonary edema, lymph node cancer(Hodgkin's disease), lymphogranulomatosis, lymphoma, lyssa, gastriccarcinoma, carcinoma of breast, meningitis, anthrax, mucoviscidosis(cystic fibrosis), multiple sclerosis (MS), myocardial infarction,neurodermatitis, neurofibromatosis, neuronal tumors, renal cancer (renalcell carcinoma), osteoporosis, pancreatic carcinoma, pneumonia,polyneuropathies, potency impairments, progressive systemic sclerosis(PSS), prostate cancer, urticaria, transverse syndrome, traumatic,rectal carcinoma, pleurisy, craniocerebral trauma, vaginal cancer(vaginal carcinoma), sinusitis, esophageal cancer, tremor, tuberculosis,tumor pain, vaginal carcinoma, burns/scalds, poisonings, viralmeningitis, menopause, soft tissue sarcoma, soft tissue tumor,impairments of cerebral blood flow and/or CNS tumors.

A cancer or tumor that is treated or prevented according to thisdisclosure maybe selected from the group of cancers or neoplasticdiseases of the ear/nose/throat region, of the lung, of the mediastinum,of the gastrointestinal tract, of the urogenital system, of thegynecological system, of the breast, of the endocrine system, of theskin, bone and soft tissue sarcomas, mesotheliomas, melanomas, neoplasmsof the central nervous system, cancers or neoplastic diseases inchildhood, lymphomas, leukemias, paraneoplastics syndromes, metastaseswithout known primary tumor (CUP syndrome), peritoneal carcinomatoses,immunosuppression-related malignancies and/or tumor metastases.

The tumors may be one of the following types of cancer: adenocarcinomaof the breast, of the prostate and of the colon; all types of lungcancer derived from the bronchia; bone marrow cancer, melanoma,hepatoma, neuroblastoma; papilloma; apudoma, choristoma, branchioma;malignant carcinoid syndrome; carcinoid heart disease; carcinoma (forexample Walker's carcinoma, basal cell carcinoma, basosquamouscarcinoma, Brown-Pearce carcinoma, ductal carcinoma, Ehrlich's tumor,carcinoma in situ, Krebs-2carcinoma, Merkel cell carcinoma, mucouscarcinoma, non-small cell bronchial carcinoma, oat cell carcinoma,papillary carcinoma, scirrhous carcinoma, bronchiole-alveolar carcinoma(bronchial carcinoma, squamous cell carcinoma and transitional cellcarcinoma); histiocytic dysfunction; leukemia (for example in connectionwith B-cell leukemia, mixed cell leukemia, null cell leukemia, T-cellleukemia, chronic T-cell leukemia, HTLV II-associated leukemia, acutelymphocytic leukemia, chronic lymphocytic leukemia, master cell leukemiaand myeloid leukemia); malignant histiocytosis, Hodgkin's disease,non-Hodgkin lymphoma, solitary plasma cell tumor; reticuloendotheliosis,chondroblastoma; chondroma, chondrosarcoma; fibroma; fibrosarcoma; giantcell tumors; histiocytoma; lipoma; liposarcoma; leukosarcoma;mesothelioma, myxoma, myxosarcoma; osteoma; osteosarcoma; Ewing'ssarcoma; synovioma; adenofibroma; adenolymphoma; carcinosarcoma,chordoma, craniopharyngioma, dysgerminoma, hamartoma; mesenchymoma;mesonephroma, myosarcoma, ameloblastoma, cementoma; odontoma; teratoma;thymoma, chorioblastoma; adenocarcinoma, adenoma; cholangioma;cholesteatoma; cylindroma; cystadenocarcinoma, cystadenoma; granulosecell tumor; gynadroblastoma; hidradenoma; islet cell tumor; leydig celltumor; papilloma, sertoli cell tumor, theka cell tumor, leiomyoma;leiomyosarcoma; myoblastoma; myoma; myosarcoma; rhabdomyoma;rhabdomyosarcoma; ependynoma; ganglioneuroma, glioma; medulloblastoma,meningioma; neurilemmonoma; neuroblastoma; neuroepithelioma,neurofibrona, neuroma, paraganglioma, non-chromaffin paraganglioma,angiokeratoma, angiolymphoid hyperplasia with eosinophilia; sclerosierenangiomas; angiomatosis; glomangioma; hemangioendothelioma; hemangioma;hemangiopericytoma, hemangiosarcoma; lymphangioma, lymphangiomyoma,lymphangiosarcoma; pinealoma; cystosarcoma phyllodes; hemangiosarcoma;lymphangiosarcoma; myxosarcoma, ovarian carcinoma; sarcoma (for exampleEwing's sarcoma, experimentally, caposi sarcoma and master cellsarcoma); neoplasms (for example bone neoplasms, breast neoplasms,neoplasms of the digestive system, colorectal neoplasms, liverneoplasms, pancreatic neoplasms, pituitary neoplasms, testicularneoplasms, orbital neoplasms, neoplasms of the head and neck, of thecentral nervous system, neoplasms of the hearing organ, of the pelvis,of the respiratory tract and of the urogenital tract); neurofibromatosisand cervical squamous cell dysplasia.

The cancer or the tumor that is treated or prevented maybe tumors of theear nose and throat region including tumors of the inner nose, of theparanasal sinuses, of the nasopharynx, of the lips, of the oral cavity,of the oropharynx, of the larynx, of the hypopharynx, of the ear, of thesalivary glands and paragangliomas, tumors of the lung includingnon-small cell bronchial carcinomas, small cell bronchial carcinomas,tumors of the mediastinum, tumors of the gastrointestinal tractincluding tumors of the esophagus, of the stomach, of the pancreas, ofthe liver, of the gall bladder and of the biliary tract, of the smallbowel, colon and rectal carcinomas and anal carcinomas, urogenitaltumors including tumors of the kidneys, of the uretas, of the bladder,of the prostate, of the urethra, of the penis and of the testis,gynecological tumors including tumors of the cervix, of the vagina, ofthe vulva, corpus carcinoma, malignant trophoblastic disease, ovariancarcinoma, tumors of the uterine tube (tuba faloppii), tumors of theabdominal cavity, carcinomas of the breast, tumors of endocrine organsincluding tumors of the thyroid, of the parathyroid, of the adrenalcortex, endocrine pancreatic tumors, carcinoid tumors and carcinoidsyndrome, multiple endocrine neoplasms, bone and soft tissue sarcomas,mesotheliomas, skin tumors, melanomas including cutaneous andintraocular melanomas, tumors of the central nervous system, tumours inchildhood including retinoblastoma, Wilm's tumor, neurofibromatosis,neuroblastoma, Ewing's sarcoma tumor family, rhabdomyosarcoma, lymphomasincluding non-Hodgkin lymphomas, cutaneous T-cell lymphomas, primarylymphomas of the central nervous system, Hodgkin's disease, leukemiasincluding acute leukemias, chronic myeloid and lymphatic leukemias,plasma cell neoplasms, myelodysplastic syndromes, paraneoplasticsyndromes, metastases without known primary tumor (CUP syndrome),peritoneal carcinomatosis, immunosuppression-related malignancyincluding AIDS-related malignancies such as Kaposi's sarcoma,AIDS-associated lymphomas, AIDS-associated lymphomas of the centralnervous system, AIDS-associated Hodgkin's disease and AIDS-associatedanogenital tumors, transplantation-related malignancies, metastatictumors including brain metastases, lung metastases, liver metastases,bone metastases, pleural and pericardial metastases and malignantascites.

The cancer or the tumor that is treated or prevented may also beselected from cancers or neoplastic diseases of the carcinomas of thebreast, of the gastrointestinal tumors, including colon carcinomas,gastric carcinomas, pancreatic carcinomas, colon cancer, small bowelcancer, of the ovarian carcinomas, of the cervical carcinomas, lungcancer, prostate cancer, renal cell carcinomas and/or liver metastases.

Immunopathological reactions cause the loss of function of a particulartissue and may destroy tissues. The use according to the inventionallows active regulatory Th cells to be isolated from patients withthese diseases in order to treat the tissue-damaging immune response. Inthe case of neoplastic diseases, the use according to the invention isintended to make it possible to specifically eliminate tumor-specificTreg in vitro or ex vivo. If the activated regulatory Th cells arepresent in too small a number, they can be separated and concentratedand/or grown in cell cultures. In both cases, the patient could betreated with the concentrated and/or grown and thus increased number ofregulatory Th cells, which would lead to the expectation of suppressionof the immunopathological response and thus an improvement and/or cureof the disease. Autoimmune diseases for which CD137 could be used asmarker for identifying and/or isolating and subsequently concentratingactive regulatory Th cells and would be expected to be a successfultherapy include, but are not restricted only thereto, diabetes mellitus(IDDM), multiple sclerosis (MS), atherosclerosis (AS), psoriasis,inflammations of the bowel, autoimmune-related hemolytic anemia,Sjogren's syndrome, autoimmune-related thyroiditis and systemic lupuserythematosis.

Regulatory Th cells maybe able in connection with transplantations tosuppress an immune response, which might lead to transplant rejection.Treatment of a patient and/or subject with initially isolated and thenconcentrated specifically activated regulatory Th cells might preventand/or impede transplant rejection. Transplant rejections are induced byan immune response of the transplant acceptor. In this case, the immunecells of the acceptor attack the exogenous tissue. In this form of use,Th cells of the target patient would be exposed in vitro to cells, cellextracts, isolated antigens or proteins of the organ donor before orafter the transplantation. After this, the active regulatory Th cellswould, after an appropriate growing time, be identified and/or separatedby means of the CD137 marker. The regulatory Th cells identified and/orseparated by using the method of the invention would then be returned to(i.e., administered to) the transplant acceptor. The result thereof isthat the cells treated by this method suppress the immune response tothe transplant, and the transplant rejection is delayed or prevented.Use of the CD137 marker might be employed inter alia for allogeneictransplants and/or heterotransplants such as, for example, fortransplantations of the heart, lungs, bowel, cornea, kidneys and bonemarrow.

In the case of an allergy, regulatory Th cells would be identifiedand/or separated either before the polyclonal activation of the T cellsor after culturing with specific allergens. The identified and/orseparated cells would then be returned, after a possible propagation, tothe patient. It is then expected that the regulatory Th cells treated inthis way will suppress the immune response, and the symptoms correlatingwith the antigens used will improve.

The treatment of allergies by means of identifying and/or isolatingactive regulatory Th cells with the CD137 marker and the describedmethod would be suitable inter alia for skin rashes, atopic dermatitis,asthma, allergic rhinitis, insect toxins and food allergy such as, forexample, toward gluten, dairy products, nuts and/or fish antigens.

In the case of infectious diseases it is important to strengthen theimmune response rather than to weaken it. In certain diseases it isappropriate to modulate the immune response in order to alter theprogression of the disease. Such cases of modulation occur in diseaseswhere immunopathological reactions occur through the infection anddestroy tissue, and in those where the immune response does not protectthe body. It would be possible through the identification and/orisolation of active regulatory Th cells to suppress the immune responseand ameliorate and/or prevent immunopathological damage.

A further alternative would be treatment with regulatory Th cells, whichhave been isolated from a patient and exposed to particular antigens inorder to obtain regulatory Th cells which could redirect the immuneresponse in a defined, particular direction.

Genes can be introduced into the cells before culturing ortransplantation for a diversity of purposes, e.g. in order to prevent orreduce the susceptibility to an infection, to replace genes which aresubject to a loss of a functional mutation, to increase the ability ofthe Treg to inhibit Th cells etc. It is moreover possible to introducevectors, which express antisense mRNA or ribozymes, thus blocking theexpression of an unwanted gene. Other methods of gene therapy are theintroduction of drug-resistance genes, for example the drug-resistancegene (MDR) or antiapoptosis genes such as bcl-2. A further possibilityis to use techniques known to the skilled worker for transfecting targetcells, e.g. by electroporation, calcium-precipitated DNA, fusion,transfection or lipofection. The particular way in which the DNA isintroduced is not crucial for use of the invention.

Many vectors can be used in order to transfer exogenous genes intomammalian cells. The vectors may be episomal, e.g. plasmids, virusvectors such as, for example, cytomegalovirus, adenovirus etc. The genescan also be integrated into the target cell genome by homologousrecombination or by random integration, such as, for example, vectorsderived from retroviruses such as MMLV, HIV-1, ALV, etc.

Active factors of the Treg cells can be analyzed by means of in vitroassays or screenings. It is also possible to carry out co-culture assaysin order to study the alterations in Tregs, which suppress themultiplication of normal T cells including CD4 T and CD8 T. Interactionswith dendritic cells and other antigen-determined cells can likewise beinvestigated. The regulatory cells separated according to the inventioncan be the starting material for a large diversity of differentanalyses, e.g. of immunoassays for protein-binding studies,determinations of cell growth, differentiation and functional activity,production of hormones etc.

EXAMPLES

The invention is described in more detail through the followingexamples, which are not limiting to the invention.

Example 1

FIG. 1: Shortly after stimulation, a large fraction of the CD137+ Tregalso expressed CD154. Peripheral Th-cells (A) were isolated using FACSor MACS from PBMC and were cultivated with allogeneic monocyte-deriveddendritic cells (moDC) for 0 to 20 hours. At the time points indicated,the expression of CD137 versus CD154 was determined usingflow-cytometry. After 8 hours, already 20% of the CD137+ T-cells alsoexpressed CD154. After 16 hours, at which the maximum CD137 expressionis detected, 30% of the cells are also positive for CD154. This showsthat, contaminating T-cells are already present at early time points,but that the maximum number of target cells expresses a marker onlyafter 16 hours. The contaminating cells can be depleted by the method ofthe invention.

Example 2

Within the CD137⁺ T-cells mixture, CD154⁻ T-cells isolated according tothe method of the invention express more FoxP3.

-   FIG. 2A: Peripheral Th-cells were isolated from PBMC using FACS or    MACS and/or cultivated for 16 hours with allogeneic monocyte-derived    dendritic cells (moDC). The expression of CD137 versus CD154 was    determined using flow-cytometry. Using FACS sorting, CD137+ cells    were separated into CD154 positive and negative cells and were    stained intercellular against FoxP3. 80% of the FoxP3 positive cells    are part of the CD137+CD154− fraction, whereas the CD137+CD154+    fraction consists to 90% of FoxP3⁻ conventional T-cells.-   FIG. 2B: Alternatively, Treg was isolated using the “MACS Treg    isolation Kit” and was stimulated polyclonally with MACSiBeads    loaded with CD3/CD28 for 6 hours. Subsequently, CD137 and CD154 as    well as the expression of FoxP3 was determined and correlated to    each other. Even after polyclonal stimulation, the CD137+CD154− Treg    contain high frequencies of FoxP3 positive Treg, whereas, e.g.,    CD154 single positive Treg show very low frequencies of FoxP3+ Treg.

Example 3

FIG. 3: Within the CD137+ T-cell mixture, only CD154+ T-cells expresseffector cytokines. T-cells expressing effector cytokines areundesirable and can be depleted with the method of the invention.Peripheral Th-cells were isolated using FACS or MACS and were cultivatedfor 16 hours with allogeneic monocyte-derived dendritic cells (moDC).The expression of CD137 versus CD154 was determined usingflow-cytometry. Using FACS, CD137+ was separated into CD154 positive andnegative cells and were stained intracellularly against IFN-gamma.

Example 4

This example shows show that the combined use of CD154 (depletingCD154+) and CD137 (enriching CD137+) allows the isolation of activatedTreg even at late timepoints of stimulation.

FIG. 4: Time window for sorting and reanalysis of CD137/CD154 double-and single-positive T-cells. Peripheral Th-cells were isolated from PBMCusing FACS or MACS and were cultivated for 16 hours with allogeneicmonocyte-derived dendritic cells (moDC). The expression of CD137 versusCD154 was determining using flow-cytometry. Using FACS, CD137+CD154−,CD137+CD154+, and CD137− CD154+ T-cells were separated and were testedin an individual suppression assay for suppressive properties (see FIG.4).

FIG. 5: Only CD154− Treg cells show suppressive properties. The sortedT-cell populations shown in FIG. 4 were co-incubated with naive CFDAmarked T-cells (1:10 Treg:naive) and was stimulated with allogeneic moDC(1:20 DC:T) for 4 to 6 days. The proliferation (CFDA loss) of the naivetarget cells is shown. In the presence of CD154− depleted Treg, theproliferation in respect to alloantigen DC is completely inhibited,whereas in the presence of CD154+ T-cells, a strong proliferationoccurs.

Example 5

FIGS. 6A to 8 demonstrate that the CD137+ T cells which co-express CD154also contain Foxp3+ T cells. However, these CD154+ Foxp3+ T cells incontrast to their CD154− counterparts do not represent true Treg withstable expression of Foxp3 as they do not express the transcriptionfactor Helios and they show no demethylation of the Foxp3 gene. Thisindicates that CD154 can be used as a discrimination marker betweenstable Treg and Treg which do not show stable regulatory phenotype andtherefore may be less useful for application in Treg based therapies.

FIGS. 6A and 6B: CD25++ FoxP3+ Treg contain CD154+ T-cells. CD4+ Tregwere sorted from PBMC using FACS based on the strong expression of CD25with a high purity (94% FoxP3+). Subsequently, the cells were stimulatedpolyclonally with PMA/ionomycin and were stained for CD137 and CD154. Ascan be seen in the figure, highly purified Treg contain up to 45% CD154+cells, which in part also expressed CD137.

FIGS. 7A, 7B and 7C: Helios is almost exclusively expressed byCD137+CD154− Treg. Helios is a transcription factor that is expressed byTreg that were generated “naturally” in the thymus but not by Treg thatare “induced” in the periphery (Thornton et al., J. Immunol. 2010184(7): 3433-41). The polyclonally activated Treg shown in FIG. 6 weresorted according to CD154 and CD137 expression (FIG. 7A) and wereexamined for the four different populations of helios mRNA (FIG. 7B).Analogously, the helios protein expression in T-cells was determined inCD137/CD154 single- or double-producers that were activated for 16 hourswith allo-DC through intracellular staining (FIG. 7C). As shown, heliosmRNA and protein is almost exclusively expressed by CD137+CD154− Treg,whereas FoxP3+CD154+ Treg express no helios.

FIG. 8: CD137+CD154− Treg show strong de-methylation of the FoxP3promoter. CD4 T-cells were stimulated with allo-DC for 16 hours andCD137 and/or CD154 single/double-positive cells were sorted. Of thesorted cells, the de-methylation of CpG motives in the promoter regionof the FoxP3 gene was analyzed (TSDR region). De-methylation in theseregions is an indication of stable expression of FoxP3 and therefore ofstable Treg identity (Baron et al Eur J Immunol. 2007 37(9):2378-89). Asshown in the figure, de-methylation of the FoxP3 promoter was foundexclusively in CD154− CD137+ Treg and not in CD154+ cells.

Example 6

FIG. 9: CD137 and CD154 are also expressed on expanded Treg from variouspopulations. Human Treg were purified from PBMC with the “CD127—TregIsolation Kit” (Miltenyi Biotec GmbH) and were expanded usingCD3/CD28-loaded magnetic particles (Treg MACSiBeads) for 14 days.Subsequently, the Treg were stimulated for the time periods indicatedusing Treg MACSiBeads and were stained for CD137 and CD154. Both, CD137and CD154 single- and double-positive Treg were identified.

Example 7

This example shows depletion of CD154+ Treg from a Treg expansionculture increases the FoxP3 purity and the long terms stability of FoxP3expression.

-   FIG. 10A: CD127− Treg were expanded for 14 days using Treg CD3/CD28    MACSiBeads and subsequently restimulated for 6 hours with Treg    MACSiBeads. After 6 hours, the MACSiBeads were removed and    subsequently, CD154+ Treg were separated through magnetic depletion    (MACS). The separated populations were then expanded in vitro for    another 6 days with Treg MACSiBeads and then FoxP3 expression was    again measured using FACS. As shown, through depletion of CD154+    Treg a) the purity of FoxP3 is increased, and b) even after repeated    expansion, a higher concentration of FoxP3 is obtained, which    indicates that non-Treg and non-stabile Treg can be separated    through CD154 depletion.-   FIG. 10B: Effect of CD154 depletion on FoxP3 purity in five    different donors. Treg of five healthy donors were expanded as    described above, and the FoxP3 expression was determined before and    after CD154 depletion. The data show that CD154 depletion is suited    for removing contaminating effector cells even from cultures of    highly purified Treg.

Example 8

Obtaining polyclonal regulatory T-cells for treatment of autoimmunity,organ transplant rejection and chronic inflammations

Regulatory T-cells can be obtained from human biological material, likeblood, PBMC, leukapheresis, tissue samples either directly or byenriching the cells with a general marker for regulatory T-cells (e.g.using CliniMACS (Miltenyi Biotec GmbH) Isolation of CD25+ cells with orwithout prior depletion of other cells (CD127+, CD8, CD19)).

The mixture thus obtained can then further be purified throughstimulation of T-cells, using all known antigens (e.g. CD3/CD28 antibodycoupled to macroscopic carrier materials, super antigens), which elicita polyclonal activation of all T-cells (the stimulation of 10⁵-5×10¹⁰cells is performed in cell culture medium, circa 10⁵-10⁷ cells per ml,at 37° C.). Stimulants are added in the usual concentrations, (e.g.magnetic particles MACSiBeads® (Miltenyi Biotec GmbH) loaded withCD3/CD28 can be used that are mixed with the T-cells in a ratio ofbetween 1:10 to 10:1) and the stimulation may last from 2 to 24 hours.

After stimulation of, for example, 2 to 24 hours, CD154 positive T-cellscan be separated from the mixture by labeling the cells, for example,using magnetic microparticles coupled to CD154 specific antibodies andremoving them from the mixture using a magnetic separation column,thereby obtaining purified activated Treg.

Additionally, markers for activated Treg can be used (e.g. CD137, latentTGF-beta (LAP), GARP (LRRC32), CD121a/b) in order to enrich subsequentlyactivated Treg further, e.g. through magnetic cell sorting or FACSsorting.

The CD154 negative or CD154 negative/CD137 positive Treg can be applieddirectly to a patient or can be expanded in a cell culture, for examplewith polyclonal T-cell stimulation methods. This procedure can berepeated at any time point of the expansion culture in order to furtherimprove the purity and to remove eventually conventional T-cells thatmight have expanded also.

Example 9

Obtaining antigen-specific Treg. A further use of the invention is thepurification of antigen-specific Treg. For this use, un-separatedT-cells, e.g. PBMC, leukapheresis, tissue samples, separated T-cells orTreg, which have been pre-enriched following another method (e.g.magnetic separated CD25+ cells), are brought into contact with aspecific antigen (proteins, peptides, antigen-loaded antigen presentingcells (APC), allogeneic APC, etc.) for 2 to 24 hours. A person of skillin the art is aware of how the bringing into contact can be performed invarious concentrations, media, and cell density. An example is theco-culture of T-cells with allogeneic APC (for example, frommonocyte-generated dendritic cells or other allogeneic dendritic cells)in a ratio of T:DC 1:1-20:1 with a cell density of between 10⁵-10⁷ cellper ml.

Subsequently, using labeling with specific antibodies that are coupledwith magnetic particles, unwanted activated conventional T-cells can beremoved by removing CD154+ cells using a magnetic separation column (forexample, CliniMACS® (Miltenyi Biotec GmbH)). Antigen-specific Treg canbe obtained with high purity through subsequent enrichment via amolecule that is specific for activated Treg (for example, CD137, latentTGF-beta (LAP), GARP (LRRC32), CD121a/b). This can also be achievedthrough labeling with specific antibodies (0.1 to 20 mg/ml) that arecoupled to magnetic microparticles, where an enrichment can be achievedagain using a magnetic separation column (for example, CliniMACS®(Miltenyi Biotec GmbH)). Alternatively, fluorochrome marked antibodiescan also be used, in which case the removal may be performed usingfluorescent activated cell sorting.

The purified Treg can be applied directly to a patient or optionallyexpanded in culture or further activated. This procedure can be repeatedat any time point in the culture or can be combined with thepurification described above after polyclonal stimulation in order toincrease the purity of the culture. Antigen-specific Treg have aselective suppressing activity on immunoreactions against these specificantigens and are therefore particularly well suited to treat allimmunoreactions (autoantigens), rejection of transplants (alloantigens)or other immune diseases involving reactions against specific antigens.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes can be made and equivalents can besubstituted without departing from the scope of the invention. Inaddition, many modifications can be made to adapt a particularsituation, material, composition of matter, process, process step orsteps, to achieve the benefits provided by the present invention withoutdeparting from the scope of the present invention. All suchmodifications are intended to be within the scope of the claims appendedhereto.

All publications and patent documents cited herein are incorporatedherein by reference as if each such publication or document wasspecifically and individually indicated to be incorporated herein byreference. Citation of publications and patent documents is not intendedas an indication that any such document is pertinent prior art, nor doesit constitute any admission as to the contents or date of the same.

1. A method of identifying activated regulatory T cells that have ademethylated Foxp3 TSDR region in a cell mixture, the method comprising:(1) obtaining a cell mixture that includes such cells and other T cells;(2) contacting the cell mixture with a first molecule that specificallybinds CD154; (3) contacting the cell mixture with a second molecule thatspecifically binds an additional marker that is different from CD154 andis specifically present on regulatory T cells; and (4) detecting cellsin the mixture that have not bound with the first molecule but havebound the second molecule, thereby identifying activated regulatory Tcells in the cell mixture that have a demethylated Foxp3 TSDR region. 2.The method of claim 1, wherein the additional marker is CD137.
 3. Themethod of claim 1, wherein the additional marker is selected from CD25,GITR, latent TGF beta (LAP), GARP (LRRC32), and CD121a/b.
 4. The methodof claim 1, wherein the first molecule and the second molecule are eachan antibody or an antibody fragment.
 5. The method of claim 1, whereinthe first molecule and the second molecule are fluorescently labeled,and cells that have not bound the first molecule but have bound thesecond molecule are detected by flow cytometry.
 6. The method of claim1, further comprising determining demethylation of the Foxp3 TSDR regionin the cells detected in step (4).
 7. The method of claim 1, furthercomprising separating cells that have not bound the first molecule buthave bound the second molecule from other cells in the cell mixture. 8.The method of claim 7, further comprising administering the separatedcells to a subject in need thereof.
 9. A method of producing an enrichedcell population of activated regulatory T cells that have a demethylatedFoxp3 TSDR region, the method comprising: (1) obtaining a cell mixturethat includes such cells and other T cells; (2) combining cells from thecell mixture with a first molecule that specifically binds CD154; (3)separating and recovering cells that have not bound the first molecule;(4) combining cells from the cell mixture with a second molecule thatspecifically binds an additional marker that is different from CD154 andis specifically present on regulatory T cells; and (5) separating andrecovering cells that have bound the second molecule; wherein thecombining the cells with the first molecule is done before, during, orafter the combining the cells with the second molecule; therebyobtaining an isolated population of activated regulatory T cells thathave a demethylated Foxp3 TSDR region.
 10. The method of claim 9,whereby CD154 positive cells are labeled with a fluorescent label, andseparation of CD154 positive cells is performed by flow cytometry. 11.The method of claim 9, comprising combining the cell mixture with boththe first molecule and the second molecule; wherein the first moleculeand the second molecule are labeled with different fluorescent labels;and separating cells from the cell mixture by fluorescence activatedcell sorting for cells that have not bound the first molecule but havebound the second molecule.
 12. The method of claim 9, whereby CD154positive cells are labeled with a magnetic microparticle, and separationof CD154 positive cells is performed using a magnetic field.
 13. Themethod of claim 9, comprising combining the cell mixture with both thefirst molecule and the second molecule; wherein the first molecule islabeled with a magnetic microparticle; and separating cells from thecell mixture that are not labeled with the microparticle but have boundthe second molecule.
 14. The method of claim 9, wherein the additionalmarker is CD137.
 15. The method of claim 9, wherein the additionalmarker is selected from CD25, GITR, latent TGF beta (LAP), GARP (LRRC32)and CD121a/b.
 16. The method of claim 9, wherein the molecule bindingCD154 is an antibody or an antibody fragment.
 17. The method of claim 9,wherein the cell mixture has been cultured in the presence of anantigen, and the isolated population is antigen specific.
 18. The methodof claim 9, wherein the cell mixture has been cultured in the presenceof a pan T cell stimulator (such as CD3/CD28 antibody), and the isolatedpopulation as a whole is not specific for any particular antigen. 19.The method of claim 9, further comprising determining whether activatedregulatory T cells obtained according to the method have a demethylatedFoxp3 TSDR region.
 20. The method of claim 9, further comprisingadministering the isolated cells to a subject in need thereof.